Master Regulatory Transcription Factor Research Articles

Master Regulatory Transcription Factors in β-Aminobutyric Acid-Induced Resistance (BABA-IR): A Perspective on Phytohormone Biosynthesis and Signaling in Arabidopsis thaliana and Hordeum vulgare.

The endogenous stress metabolite β-aminobutyric acid (BABA) primes plants for enhanced resistance against abiotic and biotic stress by activating a complex phytohormone signaling network that includes abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA), and ethylene (ET). In this study, through stringent filtering, we identify 14 master regulatory transcription factors (TFs) from the DOF, AHL, and ERF families that potentially regulate the biosynthesis and signaling of these phytohormones. Transcriptional analysis of BABA-treated Arabidopsis thaliana and Hordeum vulgare suggests that DOF family TFs play a crucial role in stress response regulation in both species. BABA treatment in A. thaliana upregulates the TFs MNB1A and PBF and enhances the expression of the genes ICS1, EDS5, and WIN3 in the SA biosynthesis pathway, potentially boosting NPR1 and PR1 in the SA signaling pathway. Conversely, in H. vulgare, the BABA-induced upregulation of TF DOF5.8 may negatively regulate SA biosynthesis by downregulating ICS1, EDS5, and PR1. Additionally, in A. thaliana, BABA triggers the expression of TF PBF, which may result in the decreased expression of MYC2, a key gene in JA signaling. In contrast, H. vulgare exhibits increased expression of ERF2 TF, which could positively regulate the JA biosynthesis genes LOX and Tify9, along with the COI1 and JAZ genes involved in the JA signaling pathway. These findings offer new perspectives on the transcriptional regulation of phytohormones during plant priming.

Extramacrochaetae regulates Notch signaling in the Drosophila eye through non-apoptotic caspase activity.

Many cell fate decisions are determined transcriptionally. Accordingly, some fate specification is prevented by Inhibitor of DNA binding (Id) proteins that interfere with DNA binding by master regulatory transcription factors. We show that the Drosophila Id protein Extra macrochaetae (Emc) also affect developmental decisions by regulating caspase activity. Emc, which prevents proneural bHLH transcription factors from specifying neural cell fate, also prevents homodimerization of another bHLH protein, Daughterless (Da), and thereby maintains expression of the Death-Associated Inhibitor of Apoptosis (diap1) gene. We found that multiple effects of emc mutations on cell growth and on eye development were all caused by reduced Diap1 levels and corresponding activation of caspases. These effects included acceleration of the morphogenetic furrow, failure of R7 photoreceptor cell specification, and delayed differentiation of non-neuronal cone cells. Within emc mutant clones, Notch signaling was elevated in the morphogenetic furrow, increasing morphogenetic furrow speed. This was associated with caspase-dependent increase in levels of Delta protein, the transmembrane ligand for Notch. Posterior to the morphogenetic furrow, elevated Delta cis-inhibited Notch signaling that was required for R7 specification and cone cell differentiation. Thus, emc mutations reveal the importance of restraining caspase activity even in non-apoptotic cells to prevent abnormal development, in the Drosophila eye through effects on Notch signaling.

Th17 pathway‐related immune signatures in the pathobiology of myasthenia gravis: Integrating the roles of regulatory/effector cytokines and transcription factors

AbstractObjectivesMyasthenia gravis (MG) is an autoimmune disorder mediated by antibodies against the acetylcholine receptor (AChR), muscle specific kinase (MuSK), and other antigens in the neuromuscular junction. Antibody production is influenced by T lymphocytes. The T helper 17 (Th17) subset, an inflammatory lineage of Th cells, is associated with several autoimmune diseases. Functional interactions between T lymphocytes and pathogenic antibody responses including aberrant Th17 cell responses have been reported in MG. However, the precise mechanism(s) underlying the activation and/or pathogenic transformation of Th17 cells are not clearly known. The current study aimed at simultaneously exploring the roles of the inducers, master regulator transcription factors, and effectors of Th17 cells in patients with MG.MethodsIn this cross‐sectional study, quantification of gene expression of IL6, IL17A, STAT3, and RORC in the peripheral mononuclear cells by quantitative polymerase chain reaction (qPCR) as well as estimation of plasma levels of IL‐1β, IL‐6, and IL‐17A cytokines by multiplex suspension assay were carried out in 59 patients with MG and 61 healthy controls.ResultsGene expression levels of IL17A were significantly upregulated in patients as compared to healthy controls. The plasma levels of IL‐1β, IL‐6, and IL‐17A were significantly elevated in patients compared to healthy controls. IL17A as well as RORC gene expressions correlated with the clinical features. IL17A gene expression levels were higher in AChR‐MG patients.ConclusionThe present study supports the crucial role of the Th17 pathway in the pathobiology of MG, including its potential influence on disease severity.

Contribution of neurons that express fruitless and Clock transcription factors to behavioral rhythms and courtship.

Animals need to integrate information across neuronal networks that direct reproductive behaviors and circadian rhythms. In Drosophila, the master regulatory transcription factors that direct courtship behaviors and circadian rhythms are co-expressed in a small set of neurons. In this study we investigate the role of these neurons in both males and females. We find sex-differences in the number of these fruitless and Clock -expressing neurons ( fru ∩ Clk neurons) that is regulated by male-specific Fru. We assign the fru ∩ Clk neurons to the electron microscopy connectome that provides high resolution structural information. We also discover sex-differences in the number of fru -expressing neurons that are post-synaptic targets of Clk -expressing neurons, with more post-synaptic targets in males. When fru ∩ Clk neurons are activated or silenced, males have a shorter period length. Activation of fru ∩ Clk neurons also changes the rate a courtship behavior is performed. We find that activation and silencing fru ∩ Clk neurons impacts the molecular clock in the sLNv master pacemaker neurons, in a cell-nonautonomous manner. These results reveal how neurons that subserve the two processes, reproduction and circadian rhythms, can impact behavioral outcomes in a sex-specific manner.

Pharmacological inhibition of RUNX1 reduces infarct size after acute myocardial infarction in rats and underlying mechanism revealed by proteomics implicates repressed cathepsin levels

Myocardial infarction (MI) results in prolonged ischemia and the subsequent cell death leads to heart failure which is linked to increased deaths or hospitalizations. New therapeutic targets are urgently needed to prevent cell death and reduce infarct size among patients with MI. Runt-related transcription factor-1 (RUNX1) is a master-regulator transcription factor intensively studied in the hematopoietic field. Recent evidence showed that RUNX1 has a critical role in cardiomyocytes post-MI. The increased RUNX1 expression in the border zone of the infarct heart contributes to decreased cardiac contractile function and can be therapeutically targeted to protect against adverse cardiac remodelling. This study sought to investigate whether pharmacological inhibition of RUNX1 function has an impact on infarct size following MI. In this work we demonstrate that inhibiting RUNX1 with a small molecule inhibitor (Ro5-3335) reduces infarct size in an in vivo rat model of acute MI. Proteomics study using data-independent acquisition method identified increased cathepsin levels in the border zone myocardium following MI, whereas heart samples treated by RUNX1 inhibitor present decreased cathepsin levels. Cathepsins are lysosomal proteases which have been shown to orchestrate multiple cell death pathways. Our data illustrate that inhibition of RUNX1 leads to reduced infarct size which is associated with the suppression of cathepsin expression. This study demonstrates that pharmacologically antagonizing RUNX1 reduces infarct size in a rat model of acute MI and unveils a link between RUNX1 and cathepsin-mediated cell death, suggesting that RUNX1 is a novel therapeutic target that could be exploited clinically to limit infarct size after an acute MI.

Transcriptional and post-translational regulation of MITF mediated by bHLH domain during the melanogenesis and melanocyte proliferation in Crassostrea gigas

Melanocyte differentiation is orchestrated by the master regulator transcription factor MITF. However, its ability to discern distinct binding sites linked to effective gene regulation remains poorly understood. This study aims to assess how co-activator acetyltransferase interacts with MITF to modulate their related lysine action, thereby mediating downstream gene regulation, including DNA affinity, stability, transcriptional activity, particularly in the process of shell pigmentation. Here, we have demonstrated that the CgMITF protein can be acetylated, further enabling selective amplification of the melanocyte maturation program. Collaboration with transcriptional co-regulator p300 advances MITF dynamically interplay with downstream targeted gene promoters. We have established that MITF activation was partially dependent on the bHLH domain, which was well conserved across species. The bHLH domain contained conserved lysine residues, including K6 and K43, which interacted with the E-box motif of downstream targeted-genes. Mutations at K6 and K43 lead to a decrease in the binding affinity of the E-box motif. CgMITF protein bound to the E-box motif within the promoter regions of the tyrosinase-related genes, contributing to melanogenesis, and also interacted with the E-box motif within the TBX2 promoter regions, associated with melanocyte proliferation. We elucidated how the bHLH domain links the transcriptional regulation and acetylation modifications in the melanocyte development in C. gigas.

Cellular reprogramming in vivo initiated by SOX4 pioneer factor activity

Tissue damage elicits cell fate switching through a process called metaplasia, but how the starting cell fate is silenced and the new cell fate is activated has not been investigated in animals. In cell culture, pioneer transcription factors mediate “reprogramming” by opening new chromatin sites for expression that can attract transcription factors from the starting cell’s enhancers. Here we report that SOX4 is sufficient to initiate hepatobiliary metaplasia in the adult mouse liver, closely mimicking metaplasia initiated by toxic damage to the liver. In lineage-traced cells, we assessed the timing of SOX4-mediated opening of enhancer chromatin versus enhancer decommissioning. Initially, SOX4 directly binds to and closes hepatocyte regulatory sequences via an overlapping motif with HNF4A, a hepatocyte master regulatory transcription factor. Subsequently, SOX4 exerts pioneer factor activity to open biliary regulatory sequences. The results delineate a hierarchy by which gene networks become reprogrammed under physiological conditions, providing deeper insight into the basis for cell fate transitions in animals.

Integrated single-cell multiomics uncovers foundational regulatory mechanisms of lens development and pathology.

Ocular lens development entails epithelial to fiber cell differentiation, defects in which cause congenital cataracts. We report the first single-cell multiomic atlas of lens development, leveraging snRNA-seq, snATAC-seq and CUT&RUN-seq to discover previously unreported mechanisms of cell fate determination and cataract-linked regulatory networks. A comprehensive profile of cis- and trans-regulatory interactions, including for the cataract-linked transcription factor MAF, is established across a temporal trajectory of fiber cell differentiation. Furthermore, we identify an epigenetic paradigm of cellular differentiation, defined by progressive loss of the H3K27 methylation writer Polycomb repressive complex 2 (PRC2). PRC2 localizes to heterochromatin domains across master-regulator transcription factor gene bodies, suggesting it safeguards epithelial cell fate. Moreover, we demonstrate that FGF hyper-stimulation in vivo leads to MAF network activation and the emergence of novel lens cell states. Collectively, these data depict a comprehensive portrait of lens fiber cell differentiation, while defining regulatory effectors of cell identity and cataract formation.

Differentiation of Acute Myeloid Leukemia Cells upon Pharmacological Inhibition of LSD1 Requires Its N-Terminal Intrinsically Disordered Region

Lysine-specific histone demethylase 1 (LSD1/KDM1A) governs hematopoiesis by regulating hematopoietic stem cell renewal and proper differentiation. Additionally, LSD1 is required for the maintenance of acute myeloid leukemia (AML) cells and has emerged as a key therapeutic target. Prior work from our group and others have revealed that the LSD1 demethylase activity is not required for AML proliferation. Instead, the interaction between LSD1 and GFI1/GFI1B is necessary to sustain AML cell survival. Thus, beyond its canonical demethylase activity, LSD1 has nonenzymatic, transcription factor (TF)-specific scaffolding functions critical for its function. Most LSD1 inhibitors function by blocking protein-protein interactions between LSD1 and TFs, even though they were originally designed as enzyme inhibitors. While studies indicate that LSD1 has important scaffolding functions, how LSD1 can coordinate with TFs to control gene expression programs is not well understood. Here, we used CRISPR-suppressor scanning to identify regions of LSD1 outside the active site that when mutated can lead to resistance to the covalent LSD1 inhibitor, GSK-LSD1. The understudied N-terminal intrinsically disordered region (IDR) of LSD1 was among the most enriched regions. To explore this further, we generated clonal AML cell lines harboring in-frame deletions within the IDR. These IDR-mutant cell lines were drug resistant and failed to differentiate upon GSK-LSD1 treatment. Moreover, knockdown of endogenous LSD1 followed by overexpression of LSD1 IDR-mutants rescued growth in the presence of GSK-LSD1, confirming that a fully functional IDR is necessary for differentiation. We performed LSD1 co-immunoprecipitation followed by mass spectrometry in wild-type and IDR-mutant cells to measure changes in LSD1 interactors upon GSK-LSD1 treatment. GSK-LSD1 treatment disrupted the LSD1-GFI1B interaction in both wild-type and mutant cells at comparable levels. Since GSK-LSD1 still induces dissociation of the mutant complex but has minimal effect on proliferation, our results suggest that the IDR-mutant cells no longer require GFI1B. In support, knockout of GFI1B inhibited growth of wild-type but not IDR-mutant cells. Strikingly, GSK-LSD1 treatment promoted LSD1's interaction with several master regulator TFs in hematopoiesis, including C/EBPα, PU.1, and RUNX1. Notably, C/EBPα was the most up-regulated interactor in both cell lines, with modest enrichment in IDR-mutant over wild-type. Using live-cell imaging assays, we found that deletions within the LSD1 IDR enhance association with these TFs. These results suggest that GSK-LSD1 reprograms LSD1-TF interactions, disrupting LSD1-GFI1B to induce LSD1-C/EBPα association, and that the IDR mutations alters this LSD1 redistribution to other TF partners. LSD1 is known to silence gene enhancers during differentiation in a process termed “enhancer decommissioning”. The mechanisms by which LSD1 occupies and controls enhancers remain elusive. To test if the LSD1 IDR regulates enhancer elements, we profiled LSD1 and H3K27ac using ChIP-seq in wild-type and IDR-mutant cells upon GSK-LSD1 treatment. Upon GSK-LSD1 treatment, disruption of LSD1-GFI1B binding was accompanied by increased LSD1 levels at other TF and H3K27ac sites, supporting the notion that loss of LSD1-GFI1B interaction induces LSD1 redistribution to other TF partner sites. A concomitant increase in H3K27ac levels was observed across LSD1 sites co-bound by C/EBPα, PU.1, or RUNX1 in wild-type cells. By contrast, in IDR-mutant cells, increases in H3K27ac levels were blocked at these sites upon GSK-LSD1 treatment, suggesting that full enhancer activation is impaired by mutations in the IDR, potentially due to altered association with master regulator TFs. This points to a function of LSD1 IDR in controlling enhancer activation for differentiation. Collectively, our study reveals a role for IDR in regulating LSD1-TF interactions, controlling enhancer activation that is necessary for AML cell differentiation. These data provide deep functional and mechanistic insights into the role of LSD1 IDR and offer important considerations for pharmacological targeting of protein-protein interactions.

Stomatal cell fate commitment via transcriptional and epigenetic control: Timing is crucial.

The formation of stomata presents a compelling model system for comprehending the initiation, proliferation, commitmentand differentiation of de novo lineage-specific stem cells. Precise, timelyand robust cell fate and identity decisions are crucial for the proper progression and differentiation of functional stomata. Deviations from this precise specification result in developmental abnormalities and nonfunctional stomata. However, the molecular underpinnings of timely cell fate commitment have just begun to be unravelled. In this review, we explore the key regulatory strategies governing cell fate commitment, emphasizing the distinctions between embryonic and postembryonic stomatal development. Furthermore, the interplay of transcription factors and cell cycle machineries is pivotal in specifying the transition into differentiation. We aim to synthesize recent studies utilizing single-cell as well as cell-type-specific transcriptomics, epigenomicsand chromatin accessibility profiling to shed light on how master-regulatory transcription factors and epigenetic machineries mutually influence each other to drive fate commitment and maintenance.

Characterizing Different Modes of Interplay Between Rap1 and H3 Using Inducible H3-depletion Yeast

Histones and transcription factors (TFs) are two important DNA-binding proteins that interact, compete, and together regulate transcriptional processes in response to diverse internal and external stimuli. Condition-specific depletion of histones in Saccharomyces cerevisiae using a galactose-inducible H3 promoter provides a suitable framework for examining transcriptional alteration resulting from reduced nucleosome content. However, the effect on DNA binding activities of TFs is yet to be fully explored. In this work, we combine ChIP-seq of H3 with RNA-seq to elucidate the genome-scale relationships between H3 occupancy patterns and transcriptional dynamics before and after global H3 depletion. ChIP-seq of Rap1 is also conducted in the H3-depletion and control treatments, to investigate the interplay between this master regulator TF and nucleosomal H3, and to explore the impact on diverse transcriptional responses of different groups of target genes and functions. Ultimately, we propose a working model and testable hypotheses regarding the impact of global and local H3 depletion on transcriptional changes. We also demonstrate different potential modes of interaction between Rap1 and H3, which sheds light on the potential multifunctional regulatory capabilities of Rap1 and potentially other pioneer factors.

Interaction between phenylpropane metabolism and oil accumulation in the developing seed of Brassica napus revealed by high temporal-resolution transcriptomes

BackgroundBrassica napus is an important oilseed crop providing high-quality vegetable oils for human consumption and non-food applications. However, the regulation between embryo and seed coat for the synthesis of oil and phenylpropanoid compounds remains largely unclear.ResultsHere, we analyzed the transcriptomes in developing seeds at 2-day intervals from 14 days after flowering (DAF) to 64 DAF. The 26 high-resolution time-course transcriptomes are clearly clustered into five distinct groups from stage I to stage V. A total of 2217 genes including 136 transcription factors, are specifically expressed in the seed and show high temporal specificity by being expressed only at certain stages of seed development. Furthermore, we analyzed the co-expression networks during seed development, which mainly included master regulatory transcription factors, lipid, and phenylpropane metabolism genes. The results show that the phenylpropane pathway is prominent during seed development, and the key enzymes in the phenylpropane metabolic pathway, including TT5, BAN, and the transporter TT19, were directly or indirectly related to many key enzymes and transcription factors involved in oil accumulation. We identified candidate genes that may regulate seed oil content based on the co-expression network analysis combined with correlation analysis of the gene expression with seed oil content and seed coat content.ConclusionsOverall, these results reveal the transcriptional regulation between lipid and phenylpropane accumulation during B. napus seed development. The established co-expression networks and predicted key factors provide important resources for future studies to reveal the genetic control of oil accumulation in B. napus seeds.

Homeobox Gene Expression Dysregulation as Potential Diagnostic and Prognostic Biomarkers in Bladder Cancer.

Homeobox genes serve as master regulatory transcription factors that regulate gene expression during embryogenesis. A homeobox gene may have either tumor-promoting or tumor-suppressive properties depending on the specific organ or cell lineage where it is expressed. The dysregulation of homeobox genes has been reported in various human cancers, including bladder cancer. The dysregulated expression of homeobox genes has been associated with bladder cancer clinical outcomes. Although bladder cancer has high risk of tumor recurrence and progression, it is highly challenging for clinicians to accurately predict the risk of tumor recurrence and progression at the initial point of diagnosis. Cystoscopy is the routine surveillance method used to detect tumor recurrence. However, the procedure causes significant discomfort and pain that results in poor surveillance follow-up amongst patients. Therefore, the development of reliable non-invasive biomarkers for the early detection and monitoring of bladder cancer is crucial. This review provides a comprehensive overview of the diagnostic and prognostic potential of homeobox gene expression dysregulation in bladder cancer.

HOXA3 accelerates wound healing in diabetic and aged non-diabetic mammals

Chronic wounds are characterized by a persistent, hyper-inflammatory environment that prevents progression to regenerative wound closure. Such chronic wounds are especially common in diabetic patients, often requiring distal limb amputation, but occur in non-diabetic, elderly patients as well. Induced expression of HoxA3, a member of the Homeobox family of body patterning and master regulatory transcription factors, has been shown to accelerate wound closure in diabetic mice when applied topically as a plasmid encased in a hydrogel. We now provide independent replication of those foundational in vivo diabetic wound closure studies, observing 16% faster healing (3.3 mm wounds vs 3.9 mm wounds at Day 9 post original injury of 6 mm diameter) under treatment with observable microscopic benefits. We then expand upon these findings with minimal dose threshold estimation of 1 μg HoxA3 plasmid delivered topically at a weekly interval. Furthermore, we observed similarities in natural wound healing rates between aged non-diabetic mice and young diabetic mice, which provided motivation to test topical HoxA3 plasmid in aged non-diabetic mice. We observed that HoxA3 treatment achieved complete wound closure (0 mm diameter) at 2 weeks whereas untreated wounds were only 50% closed (3 mm wound diameter). We did not observe any gross adverse effects macroscopically or via histology in these short studies. Whether as a plasmid or future alternative modality, topical HoxA3 is an attractive translational candidate for chronic wounds.

Unpacking the Complexity of Epithelial Plasticity: From Master Regulator Transcription Factors to Non-Coding RNAs.

Cellular plasticity in cancer enables adaptation to selective pressures and stress imposed by the tumor microenvironment. This plasticity facilitates the remodeling of cancer cell phenotype and function (such as tumor stemness, metastasis, chemo/radio resistance), and the reprogramming of the surrounding tumor microenvironment to enable immune evasion. Epithelial plasticity is one form of cellular plasticity, which is intrinsically linked with epithelial-mesenchymal transition (EMT). Traditionally, EMT has been regarded as a binary state. Yet, increasing evidence suggests that EMT involves a spectrum of quasi-epithelial and quasi-mesenchymal phenotypes governed by complex interactions between cellular metabolism, transcriptome regulation, and epigenetic mechanisms. Herein, we review the complex cross-talk between the different layers of epithelial plasticity in cancer, encompassing the core layer of transcription factors, their interacting epigenetic modifiers and non-coding RNAs, and the manipulation of cancer immunogenicity in transitioning between epithelial and mesenchymal states. In examining these factors, we provide insights into promising therapeutic avenues and potential anti-cancer targets.

Characterization of radiation-resistance mechanism in Spirosoma montaniterrae DY10T in terms of transcriptional regulatory system

To respond to the external environmental changes for survival, bacteria regulates expression of a number of genes including transcription factors (TFs). To characterize complex biological phenomena, a biological system-level approach is necessary. Here we utilized six computational biology methods to infer regulatory network and to characterize underlying biologically mechanisms relevant to radiation-resistance. In particular, we inferred gene regulatory network (GRN) and operons of radiation-resistance bacterium Spirosoma montaniterrae DY10^T and identified the major regulators for radiation-resistance. Our results showed that DNA repair and reactive oxygen species (ROS) scavenging mechanisms are key processes and Crp/Fnr family transcriptional regulator works as a master regulatory TF in early response to radiation.

Cell Cycle Dynamics during Stomatal Development: Window of MUTE Action and Ramification of Its Loss-of-Function on an Uncommitted Precursor.

Plants develop in the absence of cell migration. As such, cell division and differentiation need to be coordinated for functional tissue formation. Cellular valves on the plant epidermis, stomata, are generated through a stereotypical sequence of cell division and differentiation events. In Arabidopsis, three master regulatory transcription factors, SPEECHLESS (SPCH), MUTE and FAMA, sequentially drive initiation, proliferation and differentiation of stomata. Among them, MUTE switches the cell cycle mode from proliferative asymmetric division to terminal symmetric division and orchestrates the execution of the single symmetric division event. However, it remains unclear to what extent MUTE regulates the expression of cell cycle genes through the symmetric division and whether MUTE accumulation itself is gated by the cell cycle. Here, we show that MUTE directly upregulates the expression of cell cycle components throughout the terminal cell cycle phases of a stomatal precursor, not only core cell cycle engines but also check-point regulators. Time-lapse live imaging using the multicolor Plant Cell Cycle Indicator revealed that MUTE accumulates up to the early G2 phase, whereas its successor and direct target, FAMA, accumulate at late G2 through terminal mitosis. In the absence of MUTE, meristemoids fail to differentiate and their G1 phase elongates as they reiterate asymmetric divisions. Together, our work provides the framework of cell cycle and master regulatory transcription factors to coordinate a single symmetric cell division and suggests a mechanism for the eventual cell cycle arrest of an uncommitted stem-cell-like precursor at the G1 phase.

Epigenomic integrative analysis pinpoint master regulator transcription factors associated with tumorigenesis in squamous cell carcinoma of oral tongue.

Head and Neck Cancer (HNC) is a heterogeneous group of cancers, which includes cancers arising in the oral cavity, nasopharynx, oropharynx, hypopharynx, and larynx. Epidemiological studies have revealed that several factors such as tobacco and alcohol use, exposure to environmental pollutants, viral infection, and genetic factors are risk factors for developing HNC. The squamous cell carcinoma of oral tongue (SCCOT), which is significantly more aggressive than the other forms of oral squamous cell carcinoma, presents a propensity for rapid local invasion and spread, and a high recurrence rate. Dysregulation in the epigenetic machinery of cancer cells might help uncover the mechanisms of SCOOT tumorigenesis. Here, we used DNA methylation changes to identify cancer-specific enhancers that were enriched for specific transcription factor binding sites (TFBS), and potential master regulator transcription factors (MRTF) associated with SCCOT. We identified the activation of MRTFs associated with increased invasiveness, metastasis, epithelial-to-mesenchymal transition, poor prognosis, and stemness. On the other hand, we found the downregulation of MRTFs associated with tumor suppression. The identified MRTFs should be further investigated to clarify their role in oral cancer tumorigenesis and for their potential use as biological markers.

Dissecting the dynamic transcriptional landscape of early T helper cell differentiation into Th1, Th2, and Th1/2 hybrid cells.

Selective differentiation of CD4+ T helper (Th) cells into specialized subsets such as Th1 and Th2 cells is a key element of the adaptive immune system driving appropriate immune responses. Besides those canonical Th-cell lineages, hybrid phenotypes such as Th1/2 cells arise in vivo, and their generation could be reproduced in vitro. While master-regulator transcription factors like T-bet for Th1 and GATA-3 for Th2 cells drive and maintain differentiation into the canonical lineages, the transcriptional architecture of hybrid phenotypes is less well understood. In particular, it has remained unclear whether a hybrid phenotype implies a mixture of the effects of several canonical lineages for each gene, or rather a bimodal behavior across genes. Th-cell differentiation is a dynamic process in which the regulatory factors are modulated over time, but longitudinal studies of Th-cell differentiation are sparse. Here, we present a dynamic transcriptome analysis following Th-cell differentiation into Th1, Th2, and Th1/2 hybrid cells at 3-h time intervals in the first hours after stimulation. We identified an early bifurcation point in gene expression programs, and we found that only a minority of ~20% of Th cell-specific genes showed mixed effects from both Th1 and Th2 cells on Th1/2 hybrid cells. While most genes followed either Th1- or Th2-cell gene expression, another fraction of ~20% of genes followed a Th1 and Th2 cell-independent transcriptional program associated with the transcription factors STAT1 and STAT4. Overall, our results emphasize the key role of high-resolution longitudinal data for the characterization of cellular phenotypes.

Abstract 6069: HOXA9 overexpression contributes to stem cell overpopulation that drives development and growth of colorectal cancer

Abstract HOX genes encode master regulatory transcription factors that regulate stem cells (SCs), but their role in carcinogenesis is not fully understood. SC overpopulation drives colorectal cancer (CRC) development, which is also linked with the aberrant expression of specific HOX genes. One such gene is HOXA9. Our goal is to find out how HOXA9-regulatory mechanisms play a role in the SC origin of CRC. Previous research in our lab reported that HOXA9 as well as other HOX proteins (e.g. HOXA4 & HOXD10) are selectively expressed in human colonic cancer SCs (CSCs). The SC marker aldehyde dehydrogenase (ALDH) and other retinoic acid (RA) pathway components are also selectively expressed in CSCs. Moreover, HOXA9 expression is positively correlated with expression of ALDH genes such as ALDH1A1. These findings suggest that RA signaling regulates HOXA9 gene expression in colonic ALDH+ SCs. Hypothesis: Dysregulated HOXA9 expression leads to ALDH+ SC overpopulation, which contributes to CRC growth and development. We used immunofluorescent (IF) mapping to determine the histologic location of HOXA9+ cells in relation to colonic SCs and other differentiated colonic cell types in normal and neoplastic human colonic tissues. We also measured the effects of all-trans retinoic acid (ATRA) and the ALDH enzyme inhibitor diethylamino benzaldehyde (DEAB) on HOXA9 expression, cell proliferation and differentiation using CRC cell lines. Immunofluorescence mapping indicated that not only is HOXA9 selectively expressed in ALDH-positive SCs, but that HOXA9 expression is increased in colon carcinoma tissue compared to normal colon epithelium. Our results also showed that both ATRA and DEAB treatment decreased the proliferation of HT29 and SW480 CRC cell lines. Western blot analysis showed that HOXA9 has reduced expression in ATRA-treated HT29 CRC cells. Additionally, our independent bioinformatics analyses show that HOXA9 is predominantly expressed in both stem cells and neuroendocrine cells (NECs) in normal human colonic epithelium. Taken together, these findings indicate that regulation of HOXA9 gene expression by RA signaling controls differentiation of ALDH+ SCs along the NEC lineage in human colon tissue. Thus, our results show that HOXA9 is regulated by RA signaling and when dysregulated, contributes to CRC development and growth. These findings provide a mechanism that may be targeted in the design of novel treatments against SC overpopulation in CRC. Citation Format: Brian T. Osmond, Caroline O. Facey, Lynn M. Opdenaker, Bruce M. Boman. HOXA9 overexpression contributes to stem cell overpopulation that drives development and growth of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6069.